一株抗菌海洋放线菌的分离鉴定及其活性成分的初步研究
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摘要
本研究以辽宁、山东、浙江、福建、广西等地采集的海泥样品中分离、纯化获得的放线菌为供试菌,嗜水气单胞菌(Aeromonas hydrophila,编号ST78-3-3)、肠型点状产气单胞菌(Aeromonas punctata f.instestinalis,编号XP91-4-1)、鳗弧菌(Vibrioanguillarum,编号E-3-11)、杀鲑气单胞菌(Aeromonas salmonida)、哈维弧菌(Vibrioharvey)、大肠杆菌(Eschenchia coli)和金黄葡萄球菌(Staphlococcus aureus)为指示菌,采用琼脂扩散法和平板孔阱扩散法,筛选出一株具有抗菌谱广、活性较强的放线菌FA-F-5。通过多相分类法鉴定、水产动物疾病治疗试验、对鲫鱼免疫功能的影响试验、FA-F-5发酵条件优化试验、抗菌活性物质稳定性试验以及对抗菌活性物质的初步分离进行了研究,得到如下结论:
1 FA-F-5的分离鉴定:通过对FA-F-5的16S rDNA序列测定,与GenBank数据库中的16S rDNA序列进行比对,其同弗氏链霉菌(Streptomyces fradiae)的相似率达到99.5%;系统进化树分析表明二者之间的进化距离相隔较近;结合放线菌FA-F-5的形态特征、培养特征及生理生化特性,将其确定为弗氏链霉菌(Streptomyces fradiae)。
2 FA-F-5发酵液生物治疗试验:以嗜水气单胞菌感染健康鲫鱼,投喂含有抗菌活性物质的药饵,当饲料中抗菌物质用量达到370μg/kg·d时,鲫鱼没有发病,解剖内脏器官划平板无嗜水气单胞菌,证明该活性物质在生物体内具有较好的抗菌效果;分别以最佳治愈剂量的5倍量和10倍量饲喂鲫鱼,其内脏和体表未表现任何毒理现象,说明该活性物质对鲫鱼无急性毒性。
3 FA-F-5发酵液对鲫鱼免疫功能影响试验:FA-F-5发酵液经浓缩制饵投喂鲫鱼,连续投喂14d,分别于0d,7d,14d检测鲫鱼吞噬细胞杀菌活性和血清凝集抗体效价,以研究该抗菌活性成分对鲫鱼免疫功能的影响。结果表明,试验组吞噬细胞杀菌活性和血清凝集抗体效价与对照组相比存在显著性差异(P＜0.01),证明该抗菌活性成分能增强鲫鱼的免疫功能。
4 FA-F-5发酵优化试验:应用正交试验对海洋放线菌FA-F-5的发酵条件进行优化,确定其最佳培养基配方为:麦芽糖15g/L、黄豆粉25g/L、CaCO_3 1g/L、KH_2PO_40.5g/L、MgSO_4·7H_2O 0.5g/L、海盐10 g/L,最佳发酵条件为:发酵培养基初始pH 7、发酵温度为25℃、种子液菌龄为48 h、接种量为125 mL/L、装样量为100 mL/L、发酵时间为96h。最佳发酵条件下发酵液抗菌活性相对于庆大霉素和环丙沙星的效价分别为为2815IU/mL和61.43 mg/L。
5 FA-F-5抗菌活性成分稳定试验:分别对发酵液进行不同温度、pH、光照强度、储存时间等处理,测定处理前后发酵液的抗菌活性的变化。试验结果证实在处理前后发酵液中的抗菌活性成分的抑菌圈直径未发生变化,说明其具有较强的热稳定性、酸碱稳定性,光稳定性和贮藏稳定性。
6抗菌活性物质的初步分离纯化:FA-F-5发酵液经离心浓缩、732型树脂吸附、聚酰胺柱层析、醇沉,获得抗生素粗品。经碘化铋钾薄层显色试验证明该抗生素粗品属于氨基环醇类抗生素。
本试验获得的具有抗菌活性的放线菌,对于微生物资源的开发利用具有重要意义。同时对海洋放线菌FA-F-5分类地位、发酵液生物有效性、发酵条件优化及活性物质的初步分离纯化进行了初步研究,以期为开发水产专用的放线菌生物渔药提供基础资料。
This thesis was aimed to isolate actinomycetes from the sea mud samples,which came from Liaoning,Shandong,Zhejiang,Fujian and Guangxi province.To screen strains producing antibacterial product,we use agar diffusion method and flat hole-trap method against Aeromonas hydrophila,Aeromonas punctata f.instestinalis,Vibrio anguillarum,Aeromonas salmonida,Vibrio Harvey,Eschenchia coli and Staphlococcus aureus at primary screening phase,and use excavating-aperture bioassay test at examination phase.One high antibacterial strain FA-F-5 was choosen.The conclusions were gained as below by the polyphasic taxonomy,the biotherapy test,the effect of fermentation liquid on Carassius auratus' immune function and preliminary separation and purification of active component:
1 The Seperation and Identification of FA-F-5:16S rDNA gene sequence of the strain FA-F-5 was determined.The sequence was compared with 16SrDNA gene sequences in data of GenBank,the similarity between the strain FA-F-5 and Streptomyces fradiae was 99.5%.The result indicated that the strain FA-F-5 had a near system development with Streptomyces fradiae by constructing a system evolution tree of 16S rDNA.Binding morphological characteristics,cultural charachteristics and physic-biochemical charachteristics,the strain FA-F-5 was belonged to Streptomyces fradiae.
2 The Biotherapy Test of FA-F-5:The Carassius auratus infected by Aeromonas hydrophila were fed with the food containing the active component.When the dose of the active component reached 370μg/kg·d,the bacterial hemorhage disease was cured,which indicated the antibacterial effct of active component in living body was good.Meanwhile the Carassius auratus were fed with the food containing five and ten times dose of the best cural dose active component,there is no toxicological phenomenon on the surface and viscer,which indicated the active component had no acute toxicity to organism.
3 The Effect of the Condensed Fermentive Liquor on the Carassius auratus Immune Function:The Carassius auratus was fed with condensed fermentive liquor.To study the effect of the condensed fermentive liquor on the Carassius auratus' immune function,agglutinating antibody titer and bactericidal activity of phagocytes were checked on 0day,7 day and 14 day respectively.The result showed that the agglutinating antibody titer and bactericidal activity of phagocytes of the tested group was significantly different((P＜0.01) from the control group,which indicated the active component can improve the immune function of the Carassius auratus.
4 The Optimization of Fermentive Conditions:Fermentive conditions optimization test of the strain FA-F-5 was done by orthogonal design.The optimum medium for FA-F-5 was maltose 15g/L、soybean 25 g/L、、CaCO_3 1 g/L、KH_2PO_4 0.5 g/L,MgSO_4·7H_2O 0.5 g/L,salt 10 g/L. The optimum fermentive conditions was temperature 25℃,initial pH value 7,inoculation volume 125 mL/L,medium volume 100 mL/L and seed age 48 h.When the fermentive time was 96h,the strongest potency of fermentation liquid was 2158 IU/mL and 61.43μg/mL equivalenting to Gentamycin and Ciprofloxacin respectively.
5.The Stability of the Active Component:The fermentive liquor was dealed with different temperature,pH,light and storage time.The results of the stability test indicated that active substance of fermentation liquid of the strain FA-F-5 was highly stable for hyperthermy,pH,light and storage.
6 Preliminary Separation and Purification of Active Component:By means of 732 ion exchage chromatography,polyamide column chromatography and alcohol precipitation, the crude crystal of antibiotic from fermentation liquid of the strain FA-F-5 was obtained.TLC spray reagents of bismuth potassium iodide indicated the crude crystal of antibiotics belonged to the aminocyclitols antibiotic.
Some antibacterial actinomycetes were gained in this experiment,which was important means for development and utilization of microorganism resourses.Classification status of the strain FA-F-5,bioavailability of fermentation liquid,fermentive conditions optimization and initial separation and purification of active component were studied,which would settle foundation for exploration and industrialization product.
1 FA-F-5的分离鉴定:通过对FA-F-5的16S rDNA序列测定,与GenBank数据库中的16S rDNA序列进行比对,其同弗氏链霉菌(Streptomyces fradiae)的相似率达到99.5%;系统进化树分析表明二者之间的进化距离相隔较近;结合放线菌FA-F-5的形态特征、培养特征及生理生化特性,将其确定为弗氏链霉菌(Streptomyces fradiae)。
2 FA-F-5发酵液生物治疗试验:以嗜水气单胞菌感染健康鲫鱼,投喂含有抗菌活性物质的药饵,当饲料中抗菌物质用量达到370μg/kg·d时,鲫鱼没有发病,解剖内脏器官划平板无嗜水气单胞菌,证明该活性物质在生物体内具有较好的抗菌效果;分别以最佳治愈剂量的5倍量和10倍量饲喂鲫鱼,其内脏和体表未表现任何毒理现象,说明该活性物质对鲫鱼无急性毒性。
3 FA-F-5发酵液对鲫鱼免疫功能影响试验:FA-F-5发酵液经浓缩制饵投喂鲫鱼,连续投喂14d,分别于0d,7d,14d检测鲫鱼吞噬细胞杀菌活性和血清凝集抗体效价,以研究该抗菌活性成分对鲫鱼免疫功能的影响。结果表明,试验组吞噬细胞杀菌活性和血清凝集抗体效价与对照组相比存在显著性差异(P＜0.01),证明该抗菌活性成分能增强鲫鱼的免疫功能。
4 FA-F-5发酵优化试验:应用正交试验对海洋放线菌FA-F-5的发酵条件进行优化,确定其最佳培养基配方为:麦芽糖15g/L、黄豆粉25g/L、CaCO_3 1g/L、KH_2PO_40.5g/L、MgSO_4·7H_2O 0.5g/L、海盐10 g/L,最佳发酵条件为:发酵培养基初始pH 7、发酵温度为25℃、种子液菌龄为48 h、接种量为125 mL/L、装样量为100 mL/L、发酵时间为96h。最佳发酵条件下发酵液抗菌活性相对于庆大霉素和环丙沙星的效价分别为为2815IU/mL和61.43 mg/L。
5 FA-F-5抗菌活性成分稳定试验:分别对发酵液进行不同温度、pH、光照强度、储存时间等处理,测定处理前后发酵液的抗菌活性的变化。试验结果证实在处理前后发酵液中的抗菌活性成分的抑菌圈直径未发生变化,说明其具有较强的热稳定性、酸碱稳定性,光稳定性和贮藏稳定性。
6抗菌活性物质的初步分离纯化:FA-F-5发酵液经离心浓缩、732型树脂吸附、聚酰胺柱层析、醇沉,获得抗生素粗品。经碘化铋钾薄层显色试验证明该抗生素粗品属于氨基环醇类抗生素。
本试验获得的具有抗菌活性的放线菌,对于微生物资源的开发利用具有重要意义。同时对海洋放线菌FA-F-5分类地位、发酵液生物有效性、发酵条件优化及活性物质的初步分离纯化进行了初步研究,以期为开发水产专用的放线菌生物渔药提供基础资料。
This thesis was aimed to isolate actinomycetes from the sea mud samples,which came from Liaoning,Shandong,Zhejiang,Fujian and Guangxi province.To screen strains producing antibacterial product,we use agar diffusion method and flat hole-trap method against Aeromonas hydrophila,Aeromonas punctata f.instestinalis,Vibrio anguillarum,Aeromonas salmonida,Vibrio Harvey,Eschenchia coli and Staphlococcus aureus at primary screening phase,and use excavating-aperture bioassay test at examination phase.One high antibacterial strain FA-F-5 was choosen.The conclusions were gained as below by the polyphasic taxonomy,the biotherapy test,the effect of fermentation liquid on Carassius auratus' immune function and preliminary separation and purification of active component:
1 The Seperation and Identification of FA-F-5:16S rDNA gene sequence of the strain FA-F-5 was determined.The sequence was compared with 16SrDNA gene sequences in data of GenBank,the similarity between the strain FA-F-5 and Streptomyces fradiae was 99.5%.The result indicated that the strain FA-F-5 had a near system development with Streptomyces fradiae by constructing a system evolution tree of 16S rDNA.Binding morphological characteristics,cultural charachteristics and physic-biochemical charachteristics,the strain FA-F-5 was belonged to Streptomyces fradiae.
2 The Biotherapy Test of FA-F-5:The Carassius auratus infected by Aeromonas hydrophila were fed with the food containing the active component.When the dose of the active component reached 370μg/kg·d,the bacterial hemorhage disease was cured,which indicated the antibacterial effct of active component in living body was good.Meanwhile the Carassius auratus were fed with the food containing five and ten times dose of the best cural dose active component,there is no toxicological phenomenon on the surface and viscer,which indicated the active component had no acute toxicity to organism.
3 The Effect of the Condensed Fermentive Liquor on the Carassius auratus Immune Function:The Carassius auratus was fed with condensed fermentive liquor.To study the effect of the condensed fermentive liquor on the Carassius auratus' immune function,agglutinating antibody titer and bactericidal activity of phagocytes were checked on 0day,7 day and 14 day respectively.The result showed that the agglutinating antibody titer and bactericidal activity of phagocytes of the tested group was significantly different((P＜0.01) from the control group,which indicated the active component can improve the immune function of the Carassius auratus.
4 The Optimization of Fermentive Conditions:Fermentive conditions optimization test of the strain FA-F-5 was done by orthogonal design.The optimum medium for FA-F-5 was maltose 15g/L、soybean 25 g/L、、CaCO_3 1 g/L、KH_2PO_4 0.5 g/L,MgSO_4·7H_2O 0.5 g/L,salt 10 g/L. The optimum fermentive conditions was temperature 25℃,initial pH value 7,inoculation volume 125 mL/L,medium volume 100 mL/L and seed age 48 h.When the fermentive time was 96h,the strongest potency of fermentation liquid was 2158 IU/mL and 61.43μg/mL equivalenting to Gentamycin and Ciprofloxacin respectively.
5.The Stability of the Active Component:The fermentive liquor was dealed with different temperature,pH,light and storage time.The results of the stability test indicated that active substance of fermentation liquid of the strain FA-F-5 was highly stable for hyperthermy,pH,light and storage.
6 Preliminary Separation and Purification of Active Component:By means of 732 ion exchage chromatography,polyamide column chromatography and alcohol precipitation, the crude crystal of antibiotic from fermentation liquid of the strain FA-F-5 was obtained.TLC spray reagents of bismuth potassium iodide indicated the crude crystal of antibiotics belonged to the aminocyclitols antibiotic.
Some antibacterial actinomycetes were gained in this experiment,which was important means for development and utilization of microorganism resourses.Classification status of the strain FA-F-5,bioavailability of fermentation liquid,fermentive conditions optimization and initial separation and purification of active component were studied,which would settle foundation for exploration and industrialization product.
引文
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